Abstract: In this study, we investigated the pharmacological effect and possible molecular mechanism of higenamine (HG) in isoproterenol (ISO)-induced myocardial infarction (MI). All procedures were approved by the Institutional Animal Care and Use Committee of the Guangzhou University of Chinese Medicine. ISO was used to induce MI model in rats and H9c2 cells. The effects of HG on biomarkers and cardiac function in MI rats were evaluated by enzyme linked immunosorbent assay (ELISA), echocardiography and hematoxylin-eosin staining (HE). The expression of apoptosis and autophagy related proteins were detected by Western blot in myocardial tissue and H9c2 cells, as well as methyltransferase-like 3 (METTL3) and transcription factor EB (TFEB) protein expression. Molecular docking was used to evaluate the interaction between HG and METTL3. The results showed that HG significantly improved cardiac function and pathologic changes in ISO-induced MI, and inhibited the levels of MI-related biomarkers such as creatine kinase Mb (CK-MB), creatine kinase (CK) and lactate dehydrogenase (LDH). Mechanism studies showed that HG inhibited the expression of apoptosis-related proteins (Bax/Bcl2, caspase3, cleaved-caspase3). Interestingly, HG up-regulated the expression of autophagy related protein Beclin1, promoted autophagy flux, and decreased the ratio of light chain 3B-I/light chain 3B-II (LC-3B-I/LC-3B-II). Further studies found that HG increased the autophagy regulator TFEB and inhibited METTL3 expression. Molecular docking results showed that HG had a good interaction with METTL3. Taken together, HG has a potential anti-MI effect via regulating METTL3/TFEB signaling pathway-mediated autophagy.